School of Molecular Bioscience, University of Sydney, NSW 2006, Australia.
Matrix Biol. 2013 Oct-Nov;32(7-8):407-13. doi: 10.1016/j.matbio.2013.04.003. Epub 2013 Apr 15.
Elastin is predominantly comprised of crosslinked tropoelastin. For many years elastin was considered to serve a solely structural role but is now being increasingly identified as causal in cell signaling, development and repair. We introduced tropoelastin into an in vitro model in which airway smooth muscle cells (ASMCs) were stimulated with transforming growth factor (TGF)-β1 to examine the modulatory effect of this modular elastin sequence on release of angiogenic factors and matrix metalloproteinases (MMPs). Human ASMCs were presented to surfaces coated with tropoelastin or collagen and controls, then stimulated with TGF-β1. Transcript levels of vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF) were quantified 4 and 24 h after TGF-β1 stimulation. Protein VEGF release from cells and CTGF sequestered at cell surfaces were measured by ELISA at 24 and 48 h. TGF-β1 increased VEGF mRNA 2.4 fold at 4 h and 5 fold at 24 h, accompanied by elevated cognate protein release 3 fold at 24 h and 2.5 fold at 48 h. TGF-β1 stimulation increased CTGF mRNA 6.9 fold at 4 h and 11.8 fold at 24 h, accompanied by increased sequestering of its protein counterpart 1.2 fold at 24 h and 1.4 fold at 48 h. Pre-incubation of cells with tropoelastin did not modulate VEGF or CTGF mRNA expression, but combined with TGF-β1 stimulation it led to enhanced VEGF release 5.1-fold at 24h and 4.4-fold at 48 h. Pre-incubation with tropoelastin decreased CTGF sequestering 0.6-fold at 24 and 48 h, and increased MMP-2 production. Collagen pre-incubation under the same conditions displayed no effect on TGF-β1 stimulation apart from a slightly decreased (0.9 fold) sequestered CTGF at 48 h. As CTGF is known to anchor VEGF to the matrix and inhibit its angiogenic activity, a process which can be reversed by digestion with MMP-2, these findings reveal that elastin sequences can disrupt the balance of angiogenic factors, with implications for aberrant angiogenesis. The results suggest a model of molecular crosstalk and support an active role for elastin in vascular remodeling.
弹性蛋白主要由交联的原弹性蛋白组成。多年来，弹性蛋白一直被认为仅具有结构作用，但现在越来越多地被认为是细胞信号转导、发育和修复的原因。我们将原弹性蛋白引入体外模型中，其中气道平滑肌细胞 (ASMC) 受到转化生长因子 (TGF)-β1 的刺激，以研究这种模块化弹性蛋白序列对血管生成因子和基质金属蛋白酶 (MMPs) 释放的调节作用。将人 ASMC 递送至涂有原弹性蛋白或胶原蛋白和对照物的表面，然后用 TGF-β1 刺激。在 TGF-β1 刺激后 4 和 24 小时，定量测定血管内皮生长因子 (VEGF) 和结缔组织生长因子 (CTGF) 的转录水平。通过 ELISA 在 24 和 48 小时测量细胞中 VEGF 蛋白的释放和细胞表面 CTGF 的隔离。TGF-β1 在 4 小时使 VEGF mRNA 增加 2.4 倍，在 24 小时增加 5 倍，同时在 24 小时增加 3 倍，在 48 小时增加 2.5 倍。TGF-β1 刺激在 4 小时使 CTGF mRNA 增加 6.9 倍，在 24 小时增加 11.8 倍，同时其蛋白对应物的隔离增加 1.2 倍，在 48 小时增加 1.4 倍。细胞与原弹性蛋白预孵育不会调节 VEGF 或 CTGF mRNA 表达，但与 TGF-β1 刺激结合，可导致 VEGF 释放增加 5.1 倍，在 24 小时和 4.4 倍在 48 小时。原弹性蛋白预孵育可使 CTGF 隔离减少 0.6 倍，在 24 和 48 小时，同时增加 MMP-2 的产生。在相同条件下，胶原蛋白预孵育除了在 48 小时时隔离的 CTGF 略有减少（0.9 倍）外，对 TGF-β1 刺激没有影响。由于 CTGF 已知将 VEGF 锚定在基质中并抑制其血管生成活性，该过程可通过 MMP-2 消化逆转，这些发现表明弹性蛋白序列可以破坏血管生成因子的平衡，对异常血管生成有影响。结果表明存在分子串扰的模型，并支持弹性蛋白在血管重塑中的积极作用。
